This invention relates to a novel method for detecting the presence of microbial pathogens in a blood sample. In another aspect, this invention provides a novel technique for selectively separating and concentrating microbial pathogens from a blood sample. In still another aspect, this invention relates to a method for separating microbial pathogens from a lysed blood sample which may contain other components, such as antimicrobial constituents of blood and medicants present in the blood sample without the use of specialized solid or liquid filter media. In a further aspect, this invention relates to a novel method and apparatus for diagnosing septicemia.
Septicemia, which is the presence of pathogenic microorganisms in the blood, is one of the most serious types of infection encountered. Even though modern medicine has provided an armanant of antibiotics and fungal drugs, the mortality rate from septicemia is approximately 25%. In addition, when shock accompanies septicemia, the mortality rate increases to over 60%. Dibilitating diseases, major surgery, administration of immuno suppressive drugs or anticancer medication causes the patient to be particularly prone to septicemia.
Early administration of appropriate antibiotic therapy is important in fighting septicemia. Consequently, it is imperative that the physician know as rapidly as possible, not only whether the patient has septicemia, but also the identity of the infecting microorganisms and the susceptibility of the microorganisms to antibiotic agents. Thus, proper and timely diagnosis of septicemia depends upon very rapid and efficient quantitative analysis of the patient's blood. Further, it is imperative during the quantitative analysis of the patient's blood that the blood sample not be contaminated with pathogens from the laboratory environment.
Three analytical systems have been conventionally utilized to determine the presence of microorganisms in a body fluid. These conventional systems include the liquid broth culture technique, the so-called pour plate method and the filtration method using a solid matrix filter. Each of these systems has its drawbacks, and none of the systems provide for rapid detection of microorganisms in the blood sample. Generally, the liquid broth method is not quantitative, and the pour plate method and filtration method (using a solid matrix filter) are open systems subject to external contamination, e.g., the introduction of pathogens into the culture by the laboratory atmosphere or personnel.
Recently, an improved method and apparatus has been developed for determining the presence of microbial pathogenic organisms within a sample fluid including, for example, blood. This method is disclosed in U.S. Pat. No. 3,928,139, issued Dec. 23, 1975 and entitled "DETECTION OF MICROBIAL PATHOGENS". According to this improved method, rapid and quantitative detection of microbial pathogens from a sample of body fluid is provided by employing a liquid filter medium. The sample fluid is placed on a liquid filter medium within a confined sterile zone. The liquid filter medium has a density greater than the sample fluid and comprises a sterile aqueous solution which will selectively receive microbial pathogens from the sample fluid. The confined sterile zone is thereafter subjected to centrifugation to force the sample fluid against the liquid filter medium and cause microbial pathogens to selectively pass therein and thereby separate from the mass of the body fluid sample. Next, the liquid filter medium containing the microbial pathogens is separated from the remainder of the sample fluid and portions of the liquid filter medium are subjected to various culturing conditions.
The improved method described above does provide a very rapid and efficient procedure for separating microbial pathogens from a sample fluid. According to the preferred embodiment of the liquid filter medium method, the blood sample is lysed prior to the centrifugation step which causes the microbial pathogens to be selectively received by the liquid filter medium. Other pretreating agents, such as anti-coagulating agents are also used to prepare the blood sample. Some ingredients of the preferred liquid media employed by the improved method discussed above are incompatible with some of the pretreating and/or lysing agents. Furthermore, such agents will admix with the liquid filter medium if added thereto prior to the time that the blood sample is added to the confined sterile zone, and once so admixed such agents cannot diffuse from the liquid filter medium rapidly enough to effectively treat the blood. Therefore, it is necessary either to subject the blood samples to the possibility of external contamination by admixing the blood sample with the pretreating and/or lysing agents prior to introducing the sample into a closed sterile system or to employ a specialized apparatus whereby the treating agent may be contained within the closed system but separate from the liquid filter medium until the apparatus is placed into use. Apparatus of this type are disclosed in U.S. Pat. No. 3,875,012, issued Apr. 1, 1975 and entitled "APPARATUS AND METHOD FOR THE DETECTION OF MICROBIAL PATHOGENS" and in U.S. Pat. No. 3,932,222, issued Jan. 13, 1976 and entitled "FOR ISOLATING PATHOGENIC MICROORGANISMS".
Furthermore, when using the above-described improved method for detecting microbial pathogens in blood samples, using a liquid filter medium in a centrifugation vessel which includes an injectable closure means at the end of the vessel against which the blood sample and liquid filter medium are forced by centrifugation, it has been discovered that some of the heavier microbial pathogens, which are received by the liquid filter medium, can pass, under the force imparted by centrifugation, completely through the liquid filter medium and come to rest adjacent the bottom of the centrifugation vessel being employed. Unless great care is taken during separation and recovery of the liquid filter medium some of such microbial pathogens can be left behind, unrecovered. It is believed that the loss of microbial pathogens, in such cases, occurs because upon passing completely through the liquid filter medium the microbial pathogens become lodged in the tiny crevice formed between the wall of the centrifugation vessel and the injectable closure means.
In an effort to overcome the above difficulties encountered when a liquid filter media is employed a new technique has been developed employing a high density, water immiscible, hydrophobic, nontoxic, liquid cushioning agent. This technique is disclosed in applicant's copending application Ser. No. 739,274, filed Nov. 5, 1976, and entitled "METHOD FOR DETECTING MICROBIAL PATHOGENS EMPLOYING A CUSHIONING AGENT". Basically this novel technique employs a liquid substance for a cushioning agent which allows microbial pathogens to collect on or in the cushioning agent, without passing through it, upon centrifugation. The use of a liquid filter media which selectively receives the microbial pathogens, and in some cases allowed some of such pathogens to pass completely therethrough, is eliminated. Basically, the liquid cushioning agent provides a means for ensuring that separated microbial pathogens are not lost or unrecovered as a result of being entrapped in crevices or interstitial spaces within the centrifugation receptacle during the centrifugation step. This method, while being highly effective does require the use of liquid cushioning agent having specified properties.
Thus, it is desirable to have a closed, sterile method for separating and concentrating microbial pathogens suspected to be present in a blood sample without the necessity of having to premix the blood sample in a potentially contaminated environment and without the necessity of employing specially designed apparatus for accomplishing the pretreating step of the procedure. Furthermore, a procedure for recovery of microbial pathogens which does not necessitate the use of specialized filter mediums or cushioning agents is especially desirable.